Lathosterolosis,a novel multiple-malformation/mental retardation syndrome due to deficiency of 3beta-hydroxysteroid-delta5-desaturase

We report the clinical, biochemical, and molecular characterization of a patient with a novel defect of cholesterol biosynthesis. This patient presented with a complex phenotype, including multiple congenital anomalies, mental retardation, and liver disease. In the patient's plasma and cells, we found increased levels of lathosterol. The biosynthesis of cholesterol in the patient's fibroblasts was defective, showing a block in the conversion of lathosterol into 7-dehydrocholesterol. The activity of 3beta-hydroxysteroid-Delta(5)-desaturase (SC5D), the enzyme involved in this reaction, was deficient in the patient's fibroblasts. Sequence analysis of the SC5D gene in the patient's DNA, showing the presence of two missense mutations (R29Q and G211D), confirmed that the patient is affected by a novel defect of cholesterol biosynthesis.     

Pharyngeal jaw morphology and homology in Sicydiine Gobies (Teleostei: Gobiidae) and allies

An extremely large number of fifth ceratobranchial teeth, with highly modified, striated, and hooked tips were observed in the central and western Pacific sicydiine goby genus Stiphodon.A scanning electron microscopic study of the form and arrangement of fifth ceratobranchial teeth was conducted to assess the distribution of these modifications in sicydiine gobies and their putative close relatives. Our goals were to explore a new set of characters in gobioid systematics, to test sicydiine monophyly, and to test hypotheses of relationships of sicydiine gobies. Sicydiines are hypothesized herein to be most closely related to the western Pacific Tukugobius and Rhinogobius,freshwater genera with which they share thickened pelvic-fin rays, no teeth on the anterior portion of the fifth ceratobranchial bones, fifth ceratobranchial teeth with differentiated and striated tips, and overlapping anterior rami of the fifth ceratobranchial bones. The latter two characters occur in some, but not all, sicydiines. The pantropical freshwater goby Awaous,often classified with sicydiines, is not considered the closest relative of the subfamily. The highly modified fifth ceratobranchials of Stiphodon are similar to, and concluded here to be homoplasious with, those of the mudflat-dwelling New World goby Evorthodus and the Indo-west Pacific oxudercine gobies, represented in this study by Pseudapocryptes. J. Morphol. 237:257–274, 1998. Published 1998 Wiley-Liss, Inc.     

Inactivation and degradation of yeast glucose-6-phosphate dehydrogenase selectively modified by pyridoxal-5-phosphate

Modification by pyridoxal-5-phosphate of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) purified from Saccharomyces cerevisiae produces an inactivation effect, partially reversible by dilution in the presence of substrates. Spectroscopic analysis of the enzyme pyridoxal-5-phosphate complex reduced with NaBH4 provides the values expected for the binding of the aldehydic group to Lys residue. One Lys residue appears to be responsible for the observed enzyme inactivation, and the presence of the phosphate group is required for the effect. Besides the change of activity, the binding of pyridoxal-5-phosphate to the enzyme causes an increase in susceptibility to degradation by the intracellular yeast proteinase A at pH 7.6.     

Extracts from green and brown seaweeds protect tomato (Solanum lycopersicum) against the necrotrophic fungus Alternaria solani

Several reports have shown that crude or purified extracts of green, brown, and red seaweeds induce protection against fungal, bacterial, and viral pathogens in plants. In this work, we report that polysaccharide-enriched seaweed extracts obtained from green, Ulva lactuca and Caulerpa sertularioides, and brown algae, Padina gymnospora and Sargassum liebmannii, induced protection against the necrotrophic fungus Alternaria solani in tomato plants (Solanum lycopersicum). Protein activity of defense-related proteins polyphenol oxidase, guaiacol peroxidase and proteinase inhibitors together with expression levels of systemic wound response (SWRP) genes were also measured in leaf samples after algal extract treatment. All extracts were shown to reduce necrotic lesions induced by A. solani, particularly those obtained from U. lactuca and P. gymnospora. U. lactuca extracts induced the expression of SWRP genes, including defense, signal pathway, and protease genes, whereas those obtained from C. sertularioides, P. gymnospora and S. liebmannii showed almost no induction of SWRP genes, suggesting that extracts from the latter, whose carbohydrate composition varied from that of U. lactuca, may act through mechanisms other than the jasmonic acid/systemin wound-response pathway.     

Ectopic expression of the heterotrimeric G15 protein in pancreatic carcinoma and its potential in cancer signal transduction

G15 is a heterotrimeric G protein selectively expressed in immature cell lineages in adult tissues that feature higher cell renewal potential. It promiscuously couples a wide variety of G protein-coupled receptors (GPCRs) to phospholipase C. Intriguingly, G15 is poorly affected by GPCR desensitization. We show here that G15 α-subunit (Gα15) supports sustained stimulation of PKD1 by a constitutively desensitized GPCR co-transfected over a negative cell background.Based on the fact that PKD1 is a multifunctional protein kinase activated by PKC and known for promoting oncogenic signaling, we hypothesized that, if expressed out of its natural cell context, G15 might promote tumor growth. A screening for Gα15 mRNA expression pointed to pancreatic carcinoma among different human cancer cell types and revealed significant expression in human tumor biopsies xenografted in mice.In addition, G15 ectopic presence could functionally contribute to the transformation process since siRNA-induced depletion of Gα15 in pancreatic carcinoma cell lines dramatically inhibited anchorage-independent growth and resistance to the lack of nutrients.Altogether, our findings suggest that G15 supports tumorigenic signaling in pancreas and hence it may be considered as a novel potential target for the therapy of this form of cancer.     

Characterization of nitroproteome in neuron-like PC12 cells differentiated with nerve growth factor: identification of two nitration sites in alpha-tubulin

Nitric oxide (NO) is a precursor of reactive nitrating species, peroxynitrite and nitrogen dioxide, which modify proteins to generate oxidized species such as 3-nitrotyrosine that has been used as a hallmark of peroxynitrite-mediated oxidative stress on proteins. In the last few years however, a growing body of evidence indicates that NO also regulates a myriad of physiologic responses by modifying tyrosine residues. Looking for the molecular event triggered by NO in nerve growth factor (NGF)-induced neuronal differentiation, we recently reported that in differentiating PC12 cells, the cytoskeleton becomes the main cellular fraction containing nitrotyrosinated proteins, and alpha-tubulin is the major target. In the present work, we focus on the investigation of the sites of tyrosine nitration in alpha-tubulin purified by two-dimensional gel electrophoresis following anti-alpha-tubulin immunoprecipitation of protein extract from NGF-treated PC12 cells. Using Western blotting and matrix-assisted laser desorption/ionization-time of flight analysis, we show for the first time, both in vivo and in vitro, that nitration can occur on alpha-tubulin at sites other than the C-terminus and we positively identify Tyr 161 and Tyr 357 as two specific amino acids endogenously nitrated.     

Structure,regulation and evolution of the crystal-Stellate system of Drosophila

The crystal–Stellate system is one of the most known example of interaction between heterochromatin and euchromatin: a heterochromatic locus on the Y chromosome (crystal) 'represses a euchromatic locus (Stellate) on the X chromosome in Drosophila melanogaster. The molecular mechanism regulating this interaction is not completely understood. It is becoming clear that an RNA interference (RNAi) mechanism could be responsible for the silencing carried out by crystal on the Stellate sequences. Here, a detailed structural analysis of all the sequences involved in the system is reported, demonstrating a their 'puzzling structure. In addition three autosomal mutations: sting, scratch and sirio are described that interfere with the system. All of them are male sterile mutations and exhibit crystals made by the STELLATE protein in their primary spermatocytes. They are requested during oogenesis and early in embryogenesis as well. Hypothesis on the involvement of these genes in activating the Stellate sequences are discussed.     

In vitro Protein Synthesis by Plastids of Phaseolus vulgaris. II. The Probable Relation Between Ribonuclease Insensitive Amino Acid Incorporation and the Presence of Intact Chloroplasts

Amino acid incorporation into protein by chloroplasts from primary leaves of Phaseolus vulgaris L., var. Black Valentine is only partially inhibited by 400 μg/ml ribonuclease. The rate of incorporation, in the presence of ribonuclease, is progressively inhibited with time, and ceases after about half an hour. Preincubation of chloroplasts at 25°, in the absence of ribonuclease, increases the inhibitory effect of ribonuclease on the initial rate of incorporation of amino acid into protein. Examination of electron micrographs of freshly prepared chloroplast suspensions shows that chloroplasts are largely intact. However, after incubation at 25° for 1 hour the chloroplasts are disrupted, as indicated by loss of their stroma contents. It is concluded that the intact chloroplast membrane is relatively impermeable to ribonuclease. Amino acid incorporating activity probably becomes inhibited as the inside of the chloroplast is made accessible to ribonuclease by breakage of membranes during incubation at 25°.     

Trophic effects induced by alpha1D-adrenoceptors on endothelial cells are potentiated by hypoxia

Catecholamines have been shown to be involved in vascular remodeling through the stimulation of alpha(1)-adrenoceptors (alpha(1)-ARs). Recently, it has been demonstrated that catecholamines can stimulate angiogenesis in pathological conditions, even if the mechanisms and the AR subtypes involved still remain unclear. We investigated the influence of hypoxia (3% O(2)) on the ability of picomolar concentrations of phenylephrine (PHE), which are unable to induce any vascular contraction, to induce a trophic effect in human endothelial cells through stimulation of the alpha(1D)-subtype ARs. PHE, at picomolar concentrations, significantly promoted pseudocapillary formation from fragments of human mature vessels in vitro. Exposure to hypoxia significantly potentiated this effect, which was inhibited by the selective alpha(1D)-AR antagonist BMY-7378 and by the nitric oxide synthase inhibitor L-NAME, suggesting that alpha(1D)-ARs were involved in this effect through activation of the nitric oxide pathway. Proliferation and migration of HUVEC were also affected by picomolar PHE concentrations. Again, these effects were significantly potentiated in cells exposed to hypoxia and were inhibited by BMY-7378 and by N(G)-nitro-L-arginine methyl ester. Conversely, the alpha(1A)-AR-selective antagonist (S)-(+)-niguldipine hydrochloride and the alpha(1B)-AR antagonist chloroethylclonidine dihydrochloride did not modify endothelial cell migration and proliferation in response to PHE. These results demonstrate that the stimulation of alpha(1D)-ARs, triggered by picomolar PHE concentrations devoid of any contractile vascular effects, induces a proangiogenic phenotype in human endothelial cells that is enhanced in a hypoxic environment. The role of alpha(1D)-ARs may become more prominent in the adaptive responses to hypoxic vasculature injury.     

Changes in leucine transport activity in Chironomus riparius larvae after short-term exposure to potassium dichromate and fenitrothion

The effect of sublethal concentrations of potassium dichromate and fenitrothion on sodium-leucine cotransport in brush border membrane vesicles from Chironomus riparius larvae has been investigated. Exposure to potassium dichromate and fenitrothion caused a dose- and time-dependent inhibition of leucine uptake. Transport inhibition is easily detectable at doses 100-fold lower than LD50. Kinetic experiments showed that inhibition was mainly caused by a decrease of the Vmax (680 +/- 53 vs. 382 +/- 23 and 555 +/- 27 nmol/15s/mg protein in control and exposed larvae to K2Cr2O7 and fenitrothion, respectively). Inhibition is possibly related to a variation of sodium ions permeability as evidenced by increased membrane lipid peroxidation. Appropriate control experiments ruled out that the observed differences could be due to changes in general features of membrane preparations. Transport inhibition observed in larvae exposed to potassium dichromate was accompanied by changes in ascorbate peroxidase and dehydroascorbate reductase activities, whereas those exposed to fenitrothion displayed an increase in transaminase activity. The possible value of leucine uptake as biochemical biomarker is briefly discussed. Arch. Insect Biochem. Physiol. 55:90-101, 2004.